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Understanding and augmenting cancer-specific immunity is impeded by the fact that most tumors are driven by patient-specific mutations that encode unique antigenic epitopes. The shared antigens in virus-driven tumors can help overcome this limitation. Merkel cell carcinoma (MCC) is a particularly interesting tumor immunity model because (1) 80% of cases are driven by Merkel cell polyomavirus (MCPyV) oncoproteins that must be continually expressed for tumor survival; (2) MCPyV oncoproteins are only ~400 amino acids in length and are essentially invariant between tumors; (3) MCPyV-specific T cell responses are robust and strongly linked to patient outcomes; (4) anti-MCPyV antibodies reliably increase with MCC recurrence, forming the basis of a standard clinical surveillance test; and (5) MCC has one of the highest response rates to PD-1 pathway blockade among all solid cancers. Leveraging these well-defined viral oncoproteins, a set of tools that includes over 20 peptide-MHC class I tetramers has been developed to facilitate the study of anti-tumor immunity across MCC patients. Additionally, the highly immunogenic nature of MCPyV oncoproteins forces MCC tumors to develop robust immune evasion mechanisms to survive. Indeed, several immune evasion mechanisms are active in MCC, including transcriptional downregulation of MHC expression by tumor cells and upregulation of inhibitory molecules including PD-L1 and immunosuppressive cytokines. About half of patients with advanced MCC do not persistently benefit from PD-1 pathway blockade. Herein, we (1) summarize the lessons learned from studying the anti-tumor T cell response to virus-positive MCC; (2) review immune evasion mechanisms in MCC; (3) review mechanisms of resistance to immune-based therapies in MCC and other cancers; and (4) discuss how recently developed tools can be used to address open questions in cancer immunotherapy. We believe detailed investigation of this model cancer will provide insight into tumor immunity that will likely also be applicable to more common cancers without shared tumor antigens.

Identifying tumor-specific T cell clones that mediate immunotherapy responses remains challenging. Mutation-associated neoantigen (MANA) -specific CD8+ tumor-infiltrating lymphocytes (TIL) have been shown to express high levels of CXCL13 and CD39 ( ENTPD1 ), and low IL-7 receptor ( IL7R ) levels in many cancer types, but their collective relevance to T cell functionality has not been established. Here we present an integrative tool to identify MANA-specific TIL using weighted expression levels of these three genes in lung cancer and melanoma single-cell RNAseq datasets. Our three-gene “MANAscore” algorithm outperforms other RNAseq-based algorithms in identifying validated neoantigen-specific CD8+ clones, and accurately identifies TILs that recognize other classes of tumor antigens, including cancer testis antigens, endogenous retroviruses and viral oncogenes. Most of these TIL are characterized by a tissue resident memory gene expression program. Putative tumor-reactive cells (pTRC) identified via MANAscore in anti-PD-1-treated lung tumors had higher expression of checkpoint and cytotoxicity-related genes relative to putative non-tumor-reactive cells. pTRC in pathologically responding tumors showed distinguished gene expression patterns and trajectories. Collectively, we show that MANAscore is a robust tool that can greatly enrich candidate tumor-specific T cells and be used to understand the functional programming of tumor-reactive TIL. Although individual genes that distinguish tumor-reactive CD8+ T cells from bystander T cells in tumors have been described, a functionally meaningful integrative signature has not been established. Here authors show that mutation-associated neoantigen-specific CD8+ tumor-infiltrating lymphocytes can be recognized by MANAscore, an algorithm that uses weighted expression levels of CXCL13, ENTPD1 and IL7R in single-cell RNAseq datasets of lung cancer and melanoma patients as input.

Antisense technologies consist of the utilization of oligonucleotides or oligonucleotide analogs to interfere with undesirable biological processes, commonly through inhibition of expression of selected genes. This field holds a lot of promise for the treatment of a very diverse group of diseases including viral and bacterial infections, genetic disorders, and cancer. To date, drugs approved for utilization in clinics or in clinical trials target diseases other than bacterial infections. Although several groups and companies are working on different strategies, the application of antisense technologies to prokaryotes still lags with respect to those that target other human diseases. In those cases where the focus is on bacterial pathogens, a subset of the research is dedicated to produce antisense compounds that silence or reduce expression of antibiotic resistance genes. Therefore, these compounds will be adjuvants administered with the antibiotic to which they reduce resistance levels. A varied group of oligonucleotide analogs like phosphorothioate or phosphorodiamidate morpholino residues, as well as peptide nucleic acids, locked nucleic acids and bridge nucleic acids, the latter two in gapmer configuration, have been utilized to reduce resistance levels. The major mechanisms of inhibition include eliciting cleavage of the target mRNA by the host’s RNase H or RNase P, and steric hindrance. The different approaches targeting resistance to β-lactams include carbapenems, aminoglycosides, chloramphenicol, macrolides, and fluoroquinolones. The purpose of this short review is to summarize the attempts to develop antisense compounds that inhibit expression of resistance to antibiotics.

BackgroundAntibodies blocking programmed death (PD)-1 or its ligand (PD-L1) have revolutionized cancer care, but many patients do not experience durable benefits. Novel treatments to stimulate antitumor immunity are needed in the PD-(L)1 refractory setting. The stimulator of interferon genes (STING) protein, an innate sensor of cytoplasmic DNA, is a promising target with several agonists in development. However, response rates in most recent clinical trials have been low and mechanisms of response remain unclear. We report detailed biomarker analyses in a patient with anti-PD-L1 refractory, Merkel cell polyomavirus (MCPyV)-positive, metastatic Merkel cell carcinoma (MCC) who was treated with an intratumoral (IT) STING agonist (ADU-S100) plus intravenous anti-PD-1 antibody (spartalizumab) and experienced a durable objective response with regression of both injected and non-injected lesions.MethodsWe analyzed pretreatment and post-treatment tumor and peripheral blood samples from our patient with single-cell RNA sequencing, 30-parameter flow cytometry, T cell receptor sequencing, and multiplexed immunohistochemistry. We analyzed cancer-specific CD8 T cells using human leukocyte antigen (HLA)-I tetramers loaded with MCPyV peptides. We also analyzed STING expression and signaling in the tumor microenvironment (TME) of 88 additional MCC tumor specimens and in MCC cell lines.ResultsWe observed high levels of MCPyV-specific T cells (12% of T cells) in our patient’s tumor at baseline. These cancer-specific CD8 T cells exhibited characteristics of exhaustion including high TOX and low TCF1 proteins. Following treatment with STING-agonist plus anti-PD-1, IT CD8 T cells expanded threefold. We also observed evidence of likely improved antigen presentation in the MCC TME (greater than fourfold increase of HLA-I-positive cancer cells). STING expression was not detected in any cancer cells within our patient’s tumor or in 88 other MCC tumors, however high STING expression was observed in immune and stromal cells within all 89 MCC tumors.ConclusionsOur results suggest that STING agonists may be able to work indirectly in MCC via signaling through immune and stromal cells in the TME, and may not necessarily need STING expression in the cancer cells. This approach may be particularly effective in tumors that are already infiltrated by inflammatory cells in the TME but are evading immune detection via HLA-I downregulation.

BackgroundPredicting which patients will respond to PD-1 blockade and identifying the relevant underlying mechanisms are major challenges in immuno-oncology. Emerging data suggest that the frequency of cancer-specific T-cells in blood can predict anti-PD-1 response,1 2 but identifying these cells in most cancers is not routinely feasible. Prior studies have identified gene signatures shared by cancer-specific T cells within tumors, however, these cannot be applied to blood samples because gene expression patterns are strikingly different between cancer-specific T-cells in tumors versus blood. If gene signatures could be developed to identify cancer-specific T cells in blood, this could enable prediction of response. We leveraged the small antigenic space of Merkel cell polyomavirus (MCPyV) oncoproteins and our existing suite of reagents to identify and characterize cancer-specific T-cells in blood from patients with Merkel cell carcinoma (MCC).MethodsSingle-cell RNA sequencing was performed on 17 pre-anti-PD-(L)1 blood from patients with advanced MCC. MCPyV-specific CD8 T cells were identified using a panel of 19 HLA multimer reagents. Differential gene expression was used to analyze transcripts enriched in MCPyV multimer-binding cells relative to other non-naïve CD8 T-cells.ResultsWe identified a 27-gene signature that was enriched in MCPyV-specific CD8 T cells across 17 patients. An independent validation cohort of 8 virus-driven MCC patients revealed that this gene signature was able to identify peripheral MCC-associated CD8 T cells with a sensitivity of 69% and a specificity of 90% (figure 1A). We determined if this gene signature could also identify T cells from a cohort of 17 Epstein Barr Virus-driven nasopharyngeal carcinoma (NPC) patients. NPC-associated CD8 T cells were identified using a previously determined protein signature.3 Indeed, the gene signature was able to identify peripheral NPC-associated CD8 T-cells with a sensitivity of 72% and a specificity of 75% (figure 1B).ConclusionsThe tumor-specific T-cell gene signature generated via one cohort of MCC patients robustly identified tumor-specific T cells in two other cohorts (one composed of MCC patients and one of NPC patients) with comparable accuracy. Taken together, our data indicate that, compared to other non-naïve peripheral CD8 T-cells, tumor-specific CD8 T-cells have a distinct gene expression profile. It is plausible that this gene expression profile could also identify cancer-specific CD8 T-cells in mutationally driven cancers. This would allow us to gain important insights into the mechanisms of response and resistance to T-cell-based immunotherapies and enable clinically feasible identification of TCRs for transgenic T cell therapies for most cancers.Trial RegistrationThis abstract includes patient samples collected on NCT02267603.ReferencesPulliam T, Jani S, Jing L, Zhang J, Kulikauskas R, Church C, Garnett-Benson C, Paulson K, Smith K, Pardoll A, et al. 50 Merkel cell polyomavirus-specific CD8 T cells in blood, but not in tumors, correlate with immunotherapy response in merkel cell carcinoma. Journal for ImmunoTherapy of Cancer 2022;10:A53-A53. 10.1136/jitc-2022-SITC2022.0050.Ryu H, Bi T, Sarkar K, Church C, Ramchurren N, Pulliam T, Fling S, Nghiem P, Newell E. 1045 High dimensional profiling of merkel cell polyomavirus-specific T cells in response to anti-PD-1 immunotherapy. Journal for ImmunoTherapy of Cancer 2022;10:A1087-A1087. 10.1136/jitc-2022-SITC2022.1045.Kumar N, MacMillan H, Yeong JPS, Chua M, Jain A, Newell E. 1035 High-dimensional immune profiling of peripheral epstein-barr virus-specific T cells in nasopharyngeal carcinoma. Journal for ImmunoTherapy of Cancer 2022;10:A1077-A1077. 10.1136/jitc-2022-SITC2022.1035.Ethics ApprovalAll patients represented here participated with written informed consent. Training cohort samples were provided by Cancer Immunotherapy Trails Network (trial registration: ClinicalTrials.gov NCT02267603) and analyzed with approval by the Fred Hutchinson Cancer Center‘s Institutional Review Board (FH 6585). MCC validation cohort samples were collected and analyzed with approval by the Fred Hutchinson Cancer Center‘s Institutional Review Board (FH 6585). NPC validation cohort samples were obtained from the National Cancer Centre Singapore, and de-identified patient information from this cohort was obtained with approval by the institutional review board at the Fred Hutchinson Cancer Research Center (IR File#: 6007–1053).Abstract 1018 Figure 1Receiver operating characteristic (ROC) curve demonstrating performance of the tumor-specific gene expression signature in independent validation cohorts from MCC (A) and NPC (B).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hybrid immunity is more protective than vaccination or previous infection alone. To investigate the kinetics of spike-reactive T (T S ) cells from SARS-CoV-2 infection through messenger RNA vaccination in persons with hybrid immunity, we identified the T cell receptor (TCR) sequences of thousands of index T S cells and tracked their frequency in bulk TCRβ repertoires sampled longitudinally from the peripheral blood of persons who had recovered from coronavirus disease 2019 (COVID-19). Vaccinations led to large expansions in memory T S cell clonotypes, most of which were CD8 + T cells, while also eliciting diverse T S cell clonotypes not observed before vaccination. TCR sequence similarity clustering identified public CD8 + and CD4 + TCR motifs associated with spike (S) specificity. Synthesis of longitudinal bulk ex vivo single-chain TCRβ repertoires and paired-chain TCRɑβ sequences from droplet sequencing of T S cells provides a roadmap for the rapid assessment of T cell responses to vaccines and emerging pathogens. Koelle and colleagues use an activation marker-dependent approach to determine the recruitment of TCR by three doses of mRNA vaccination in individuals previously infected with SARS-CoV-2.

BackgroundPD-1 pathway blockade has revolutionized oncology, though most patients do not derive durable benefit. Accurate prediction of response is not currently possible. Cancer-specific CD8 T cells can mediate tumor regression, however, identifying these cells is difficult because many tumor antigens are patient-specific. Merkel cell carcinoma (MCC), driven by Merkel cell polyomavirus (MCPyV) oncoproteins in ~80% of cases, is an attractive cancer for studying tumor-specific T cells due to shared, viral antigens and a low tumor mutational burden. Using samples from a recent trial of neoadjuvant nivolumab in MCC (NCT02488759), we studied anti-PD-1 resistance by interrogating MCPyV-specific CD8 T cells before and during therapy.MethodsMCPyV-specific CD8 T cells were identified using an expanded panel of 16 MCPyV-specific HLA class I multimers. PBMC from 21 patients with suitable multimers (among 35 patients assessed) collected before, 2 and 4 weeks after initiating anti-PD-1 were stained with MCPyV-multimers in 26-plex flow cytometry. Intratumoral MCPyV-specific T cell frequency was calculated using a combination of 1) HLA-I multimers and paired T cell receptor (TCR)-seq of phytohemagglutinin-expanded tumor infiltrating lymphocytes to identify virus-specific TCRs and 2) beta-TCRseq of formalin-fixed tumors to determine T cell clone frequency. To study phenotypic differences between cancer-specific CD8 T cells found in tumors versus blood, single cell RNAseq and paired TCRseq were performed on a separate cohort of 7 MCC patients.ResultsPatients without detectable circulating MCPyV-specific CD8 T cells before treatment had shorter recurrence-free survival (RFS; figure 1; median RFS=12 months; n=7) than patients with detectable MCPyV-specific cells (median RFS not reached; n=11; p=0.0078). In contrast, response was not associated with intratumoral, MCPyV-specific CD8 T cells (13% [mean] of intratumoral T cells in patients with pathological complete response versus 23% in those without response; p=NS). T cells were considered ‘dysfunctional/exhausted’ if they fell within single cell RNAseq clusters characterized by TOX, PD-1, and LAG-3 transcripts. MCPyV-specific T cells were significantly more likely to be dysfunctional/exhausted if they were intratumoral (>90% dysfunctional) versus in blood (0–50%; p=0.002).ConclusionsMCC-specific CD8 T cells in blood were less dysfunctional than their intratumoral counterparts. The frequency of pre-existing MCC-specific CD8 T cells in blood strongly correlated with anti-PD-1 response, while their frequency within tumors was unrelated to response. These results suggest that approaches to increase the number of circulating, less exhausted, cancer-specific T cells may benefit patients with anti-PD-(L)1-refractory MCC, and the frequency of these cells may be a predictive marker of anti-PD-(L)1 response.Trial RegistrationNCT02488759Ethics ApprovalThis study was approved by the Fred Hutchinson Cancer Center‘s Institutional Review Board, approval number 6585. All patients represented here participated with written informed consent.Abstract 50 Figure 1T cell frequency and responseFrequency of Merkel cell polyomavirus-specific CD8 T cells in blood predicts response to neoadjuvant PD-1 pathway blockade. Kaplan Meier curve showing survival of patients who had MCPyV-specific CD8 T cells in blood above (tetramer positive; blue) or below (tetramer negative; red) the limit of detection (0.01% of CD8 T cells; p=0.0078 via log-rank test)[Figure omitted. See PDF]

External guide sequences (EGSs) are short antisense oligoribonucleotides that elicit RNase P-mediated cleavage of a target mRNA, which results in inhibition of gene expression. EGS technology is used to inhibit expression of a wide variety of genes, a strategy that may lead to development of novel treatments of numerous diseases, including multidrug-resistant bacterial and viral infections. Successful development of EGS technology depends on finding nucleotide analogs that resist degradation by nucleases present in biological fluids and the environment but still elicit RNase P-mediated degradation when forming a duplex with a target mRNA. Previous results suggested that locked nucleic acids (LNA)/DNA chimeric oligomers have these properties. LNA are now considered the first generation of compounds collectively known as bridged nucleic acids (BNAs) – modified ribonucleotides that contain a bridge at the 2ʹ,4ʹ-position of the ribose. LNA and the second-generation BNA, known as BNANC, differ in the chemical nature of the bridge. Chimeric oligomers containing LNA or BNANC and deoxynucleotide monomers in different configurations are nuclease resistant and could be excellent EGS compounds. However, not all configurations may be equally active as EGSs. RNase P cleavage assays comparing LNA/DNA and BNANC/DNA chimeric oligonucleotides that share identical nucleotide sequence but with different configurations were carried out using as target the amikacin resistance aac(6ʹ)-Ib mRNA. LNA/DNA gapmers with 5 and 3/4 LNA residues at the 5ʹ- and 3ʹ-ends, respectively, were the most efficient EGSs while all BNANC/DNA gapmers showed very poor activity. When the most efficient LNA/DNA gapmer was covalently bound to a cell-penetrating peptide, the hybrid compound conserved the EGS activity as determined by RNase P cleavage assays and reduced the levels of resistance to amikacin when added to Acinetobacter baumannii cells in culture, an indication of cellular uptake and biological activity.

The aminoglycoside 6 -N-acetyltransferase type Ib [AAC(6')-Ib] is the most widely distributed enzyme among AAC(6')-I-producing Gram-negative pathogens and confers resistance to clinically relevant aminoglycosides including amikacin. This enzyme is therefore ideal to target with enzymatic inhibitors that could overcome resistance to aminoglycosides. The search for inhibitors was carried out using mixture-based combinatorial libraries, the scaffold ranking approach, and the positional scanning strategy. A library with high inhibitory activity had pyrrolidine pentamine scaffold and was selected for further analysis. This library contained 738,192 compounds with functionalities derived from 26 different amino acids (R1, R2 and R3) and 42 different carboxylic acids (R4) in four R group functionalities. The most active compounds all contained S-phenyl (R1 and R3) and S-hydromethyl (R2) functionalities at three locations and differed at the R4 position. The compound containing 3-phenylbutyl at R4 (compound 206) was a robust enzymatic inhibitor in vitro, in combination with amikacin potentiated the inhibition of growth of three resistant bacteria in culture, and improved survival when used as treatment of Galleria mellonella infected with aac(6')-Ib-harboring Klebsiella pneumoniae and Acinetobacter baumannii strains.